Aluminum and Metal Tolerance Profiling

The growth profile of strain A7 was checked to tolerate an increasing concentration of aluminum (AlCl₃) (0–4 mM) and other metal compounds using the Single Plate Serial Dilution Spotting (SP-SDS) method on modified M9 agar plates [80 (link)]. The liquid culture was in the M9 broth supplemented with 0.1% proteases peptone. Similarly, the effect of Fe (III) from 0–5 mM, Fe (II) from 0–3 mM, NaCl (0–8%) (Sigma Aldrich, St. Louis, MO, USA) [81 (link)], pH (1–14) and temperature (4–50 °C) was checked in three biological replicates [82 (link),83 (link)]. Freshly grown bacterial cells (0.1 OD₆₀₀) were serially diluted from 10⁻¹ to 10⁻⁶, and 3 µL from each dilution was inoculated into the demarcated areas of SP-SDP and in a 10⁻¹ dilution in a 50 mL broth. The Petri dishes were incubated for 18–48 h, and the flask was held in an orbital shaker at 160 rpm at 30 ± 2 °C for 48 h. The optical density of the cultures was taken at 600 nm (PerkinElmer UV/VIS spectrometer Lamda 25, USA). The relative growth (RG) was calculated using the formula [84 (link)]. Relative Growth (%) = A_t/A_c ∗ 100, where A_c = optical density of the average of three biological replicates of controls, and A_t = optical density of average three biological replicates of the treatments. Bacterial 20% and 50% growth inhibitory concentration (IC) IC₂₀ and IC₅₀ values were calculated using the dose–response data.